Library design and screening protocol for artificial metalloenzymes based on the biotin-streptavidin technology

Abstract

Artificial metalloenzymes (ArMs) based on the incorporation of a biotinylated metal cofactor within streptavidin (Sav) combine attractive features of both homogeneous and enzymatic catalysts. To speed up their optimization, we present a streamlined protocol for the design, expression, partial purification and screening of Sav libraries. Twenty-eight positions have been subjected to mutagenesis to yield 335 Sav isoforms, which can be expressed in 24-deep-well plates using autoinduction medium. The resulting cell-free extracts (CFEs) typically contain >1 mg of soluble Sav. Two straightforward alternatives are presented, which allow the screening of ArMs using CFEs containing Sav. To produce an artificial transfer hydrogenase, Sav is coupled to a biotinylated three-legged iridium pianostool complex Cp*Ir(Biot-p-L)Cl (the cofactor). To screen Sav variants for this application, you would determine the number of free binding sites, treat them with diamide, incubate them with the cofactor and then perform the reaction with your test compound (the example used in this protocol is 1-phenyl-3,4-dihydroisoquinoline). This process takes 20 d. If you want to perform metathesis reactions, Sav is coupled to a biotinylated second-generation Grubbs-Hoveyda catalyst. In this application, it is best to first immobilize Sav on Sepharose-iminobiotin beads and then perform washing steps. Elution from the beads is achieved in an acidic reaction buffer before incubation with the cofactor. Catalysis using your test compound (in this protocol, 2-(4-(N,N-diallylsulfamoyl)phenyl)-N,N,N-trimethylethan-1-aminium iodide) is performed using the formed metalloenzyme. Screening using this approach takes 19 d.

Upregulation of an Artificial Zymogen by Proteolysis

“…5) Next, Sav mutants were screened in the presence of SGF. As previously observed, [20,26] [Cp* biot Ir(EGF)L]&S112A K121A Sav afford (R)-3b in 73 % ee and 81 % ee,respectively (Table 1e ntries 9a nd 10).…”

Streptavidin as a Scaffold for Light‐Induced Long‐Lived Charge Separation

“…The biotin‐binding capacity of the RuSav n and the RuSav n – aMV 2+ bioconjugates were evaluated relying on a displacement titration using 2‐(4‐hydroxyphenylazo)benzoic acid (HABA) . Upon incorporation within Sav, HABA displays an absorption at λ max =506 nm.…”

“…The mutantsw ere expressed using ZYP-5052 media, purifiedb ya ffinity chromatography using iminobiotinsepharosematrix and characterized by ESI-MS (details collected in the Supporting Information, Figures S10-S17). [56,57] SDS-PAGE analysisu sing biotin-4-fluorescein demonstrated the biotin binding capability of the engineered Sav tetramers and highlighted am arked changei ni ts isoelectric point pI,a se videnced by the significant difference in electrophoretic mobility ( Figure S9).…”

An Artificial Cofactor catalyzing the Baylis-Hillman Reaction using Designed Streptavidin as Protein Host

“…Especially position 121 is known to influence the outcome of metal-based catalysts to a great extent. [28] Thus, the sterically demanding Q114 was changed to A or T, and R121 and S112 were substituted by A as well. Additionally, the variant S112A, R121A was tested, but none of these variants showed activity higher than the background ( Table 1, Entry 4 and 5).…”